The epithelial sodium channel (ENaC) is of fundamental importance in the control of sodium reabsorption in the distal nephron. ENaC regulation is critical for the overall control of sodium balance and extracellular fluid volume, and thereby of blood pressure. Dysregulation of ENaC underlies some forms of essential hypertension - a common condition and a major cause of cardiovascular morbidity and mortality. However, the molecular mechanisms of ENaC regulation are not completely understood. This work aims to define the phosphorylation-dependent regulation of ENaC trafficking in renal epithelia. Nearly all research in this field has focused on the mechanisms that govern ENaC retrieval from the apical membrane. By contrast, our knowledge of the mechanisms that promote the regulated forward trafficking of ENaC to the apical cell surface is incomplete. Specifically, the phosphorylation-dependent steps and protein interactions involved in apical membrane ENaC recycling and exocytosis have not been elucidated. Based on our prior findings, 14-3-3 proteins are essential stabilizers of the phosphoproteins that regulate ENaC trafficking. Affinity capture of 14-3-3 binding proteins, combined with quantitative proteomic analysis, has given us candidates for the regulation of ENaC traffic in polarized renal epithelia. With biochemical and functional assays, we will evaluate the 14-3-3 binding phosphoproteins that impact the apical ENaC trafficking pathway. Our preliminary data has indicated that the Rab-GAP proteins, AS160 (TBC1D4) and TBC1D1, are key substrates for the protein kinases that regulate ENaC trafficking in response to aldosterone and vasopressin. To begin, we will define the mechanism of action of AS160, a newly identified 14-3-3 binding protein and phosphorylation-dependent regulator of aldosterone-mediated ENaC trafficking. Using biochemical and functional assays, we will test the hypothesis that AS160 stabilizes ENaC within intracellular compartments under basal conditions, and permits ENaC trafficking to the apical membrane in response to its phosphorylation. Second, we will determine whether TBC1D1, a new target identified by 14-3-3 affinity capture, regulates ENaC trafficking in response to vasopressin/PKA stimulation. This work is expected to reveal new mechanisms for the control of apical ENaC density, and identify novel targets for the treatment of diseases of salt and water balance in sodium transporting epithelia. PUBLIC HEALTH RELEVANCE: This study, titled Phosphorylation-dependent regulation of epithelial sodium channel (ENaC) trafficking, will investigate whether phosphorylation-dependent 14-3-3 binding proteins stabilize multiple steps in the apical ENaC trafficking pathway, and whether their binding partners, AS160 (TBC1D4) and TBC1D1, are key substrates for the protein kinases that mediate the actions of aldosterone and vasopressin. Through this study, we will advance our understanding of molecular mechanisms of ENaC regulation and the results will provide further insight into the control of sodium balance, blood volume and thereby of blood pressure.